Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

General bacillus subtillis combination expression vector for improving expression level of alpha-amylase

A Bacillus subtilis and expression vector technology, applied in the field of Bacillus subtilis integrated expression vector, can solve the problems of low production of α-amylase, degradation of α-amylase, affecting the correct folding of α-amylase, etc., so as to improve the secretion level , Improve the effect of transshipment level

Inactive Publication Date: 2016-07-13
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
View PDF2 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The production of α-amylase using the wild-type Bacillus subtilis expression system is usually very low. This is because there are many bottlenecks in the process of protein transport and secretion of α-amylase. On the one hand, when the regulatory elements involved in protein transport in the cell cannot meet the demand α-amylase cannot be transported to the appropriate cell site in time during protein transport and is degraded by intracellular proteases. On the other hand, when the limiting factors involved in protein secretion in cells are lacking, α-amylase Proper folding during secretion, resulting in degradation of α-amylase by proteases in the cell wall

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • General bacillus subtillis combination expression vector for improving expression level of alpha-amylase
  • General bacillus subtillis combination expression vector for improving expression level of alpha-amylase
  • General bacillus subtillis combination expression vector for improving expression level of alpha-amylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The first step, in the multiple cloning site of pDL (derived from BGSC, numbered ECE144) Eco RI and Bam The Bacillus subtilis Pgrac promoter (including groE promoter, lacO operon and gsiBCD ribosome binding site sequence) was introduced in the middle of HI.

[0027] Design primers, where the part in black bold font is the restriction site:

[0028] Pgrac-F: 5'-CCGGAATTCGAAAAGAATGATGTAAGC-3' (contains E co RI restriction site),

[0029] Pgrac-R: 5'-CGCGGATCCTTCCTCCTTTAATTGG-3' (contains B am HI restriction site).

[0030] Use pHT43 as a template, use the above primers to carry out PCR, and mix the components in a sterilized thin-walled centrifuge tube in the following order. The reaction system is:

[0031] Upstream primer Pgrac-F (10 μmol / L) 2 μL,

[0032] Downstream primer Pgrac-R (10 μmol / L) 2 μL,

[0033] DNA template 1 μL,

[0034] PrimeSTARMaxPremix (2×) 10 μL,

[0035] wxya 2 05 μL,

[0036] The total volume is 20 μL.

[0037] The amplification con...

Embodiment 2

[0058] The intermolecular chaperone PrsA (nucleotide sequence is SEQ ID NO: 6) can help α-amylase to quickly and correctly fold, reduce the chance of α-amylase being degraded by proteases in the cell wall, and may promote the expression and secretion of α-amylase increase in volume. The carrier of the present invention was used to evaluate the effect of PrsA overexpression on the expression and secretion of α-amylase in Bacillus subtilis.

[0059] (1) Construction of plasmid vector pPgrac-PrsA.

[0060] Based on the pPgrac-MCS plasmid, the pPgrac-PrsA integration vector was constructed.

[0061] Design primers, where the part in black bold font is the restriction site:

[0062] PrsA-F: 5'-CGGGGTACCATGAAGAAAATCGCAATAGCAGCT-3' (contains Kpn I restriction site),

[0063] PrsA-R: 5'-TGAGAGCCTTTAGCATATTATGTTGCCAACTGT-3' (contains Sac I restriction site),

[0064] Bacillus subtilis Bacillus subtilis The 168 genome is used as a template for PCR, and the components are mixed ...

Embodiment 3

[0092] Transform 1A751 and 1A751(amyE::Pgrac-PrsA) with plasmid pMA5-AmyL (AmyL gene is derived from Bacillus licheniformis CICC10181) already in the laboratory carrying the amylase gene of Bacillus licheniformis, and pick one transformant to inoculate In 2×SR medium (3% peptone, 6% yeast extract, 0.6% K 2 HPO 4 ) at 37°C overnight. On the second day, inoculate 30mL of 2×SR culture medium according to the inoculation amount of 1%, ferment at 37°C and 200rpm for 48h, centrifuge the fermented liquid at 12000rpm at 4°C for 10min, and take the supernatant to detect the amylase activity. Two parallel, the results are shown in Table 1.

[0093] The detection method of α-amylase activity refers to the national standard method (GBT24401-2009 α-amylase preparation detection method).

[0094] Definition of enzyme activity: Under specified conditions, liquefaction of 1 g of soluble starch in 1 hour is 1 unit of enzyme activity, expressed in "U / g (U / mL)". The determination method is a...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Upstream primeraaaaaaaaaa
Login to View More

Abstract

The invention discloses a general bacillus subtillis combination expression vector for improving the expression level of alpha-amylase. The invention relates to the general bacillus subtillis combination expression vector including the following parts: a bacillus subtillis promoter sequence, a multiple cloning site sequence for combining components possibly related to expression regulation, and an escherichia coli copying initiation sequence. The vector can be stably copied in escherichia coli and can be combined with bacillus subtillis chromosomes for stable expressing. After the vector is combined with bacillus subtillis chromosomes, the expression level of alpha-amylase in bacillus subtillis is obviously improved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a general-purpose Bacillus subtilis integrated expression vector, which is particularly suitable for improving the expression level of α-amylase. Background technique [0002] Bacillus subtilis ( Bacillus subtilis ) belongs to Gram-positive bacteria and is a non-pathogenic important industrial microorganism. Its whole genome sequencing work has been completed. Its genetic background and physiological properties are second only to E. coli in understanding. The use of Bacillus subtilis to express foreign proteins has many advantages: (1) high safety, and can be used in industrial production such as food and medicine; (2) can directly release secreted proteins into the medium, which is conducive to separation and purification; (3) has Efficient ability to secrete the target protein, and the eukaryotic-derived recombinant protein secreted by it can still main...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/75C12N1/21C12R1/125
Inventor 张大伟盖园明付刚
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com